zRICH, a protein induced during optic nerve regeneration in zebrafish, promotes neuritogenesis and interacts with tubulin.

Mammals do not regenerate axons in their central nervous system (CNS) spontaneously. This phenomenon is the cause of numerous medical conditions after damage to nerve fibers in the CNS of humans. The study of the mechanisms of nerve regeneration in other vertebrate animals able to spontaneously regenerate axons in their CNS is essential for understanding nerve regeneration from a scientific point of view, and for developing therapeutic approaches to enhance nerve regeneration in the CNS of humans. RICH proteins are a novel group of proteins implicated in nerve regeneration in the CNS of teleost fish, yet their mechanisms of action are not well understood. A number of mutant versions of the zebrafish RICH (zRICH) protein were generated and characterized at biochemical and cellular levels in our laboratory. With the aim of understanding the effects of RICH proteins in neuronal axon outgrowth, stable transfectants derived from the neuronal model PC12 cell line expressing zRICH Wild-Type or mutant versions of zRICH were studied. Results from differentiation experiments suggest that RICH proteins enhance neuronal plasticity by facilitating neurite branching. Biochemical co-purification results have demonstrated that zRICH binds to the cytoskeletal protein tubulin. The central domain of the protein is sufficient for tubulin binding, but a mutant version of the protein lacking the terminal domains, which cannot bind to the plasma membrane, was not able to enhance neurite branching. RICH proteins may facilitate axon regeneration by regulating the axonal cytoskeleton and facilitating the formation of new neurite branches.

Identification of a motif in BMRP required for interaction with Bcl-2 by site-directed mutagenesis studies.

Bcl-2 is an anti-apoptotic protein that inhibits apoptosis elicited by multiple stimuli in a large variety of cell types. BMRP (also known as MRPL41) was identified as a Bcl-2 binding protein and shown to promote apoptosis. Previous studies indicated that the amino-terminal two-thirds of BMRP contain the domain(s) required for its interaction with Bcl-2, and that this region of the protein is responsible for the majority of the apoptosis-inducing activity of BMRP. We have performed site-directed mutagenesis analyses to further characterize the BMRP/Bcl-2 interaction and the pro-apoptotic activity of BMRP. The results obtained indicate that the 13-17 amino acid region of BMRP is necessary for its binding to Bcl-2. Further mutagenesis of this motif shows that amino acid residue aspartic acid (D) 16 of BMRP is essential for the BMRP/Bcl-2 interaction. Functional analyses conducted in mammalian cells with BMRP site-directed mutants BMRP(13Ala17) and BMRP(D16A) indicate that these mutants induce apoptosis through a caspase-mediated pathway, and that they kill cells slightly more potently than wild-type BMRP. Bcl-2 is still able to counteract BMRP(D16A)-induced cell death significantly, but not as completely as when tested against wild-type BMRP. These results suggest that the apoptosis-inducing ability of wild-type BMRP is blocked by Bcl-2 through several mechanisms.

Deletion mutational analysis of BMRP, a pro-apoptotic protein that binds to Bcl-2.

Bcl-2 is an anti-apoptotic member of the Bcl-2 family of proteins that protects cells from apoptosis induced by a large variety of stimuli. The protein BMRP (MRPL41) was identified as a Bcl-2 binding partner and shown to have pro-apoptotic activity. We have performed deletion mutational analyses to identify the domain(s) of Bcl-2 and BMRP that are involved in the Bcl-2/BMRP interaction, and the region(s) of BMRP that mediate its pro-apoptotic activity. The results of these studies indicate that both the BH4 domain of Bcl-2 and its central region encompassing its BH1, BH2, and BH3 domains are required for its interaction with BMRP. The loop region and the transmembrane domain of Bcl-2 were found to be dispensable for this interaction. The Bcl-2 deletion mutants that do not interact with BMRP were previously shown to be functionally inactive. Deletion analyses of the BMRP protein delimited the region of BMRP needed for its interaction with Bcl-2 to the amino-terminal two-thirds of the protein (amino acid residues 1-92). Further deletions at either end of the BMRP(1-92) truncated protein resulted in lack of binding to Bcl-2. Functional studies performed with BMRP deletion mutants suggest that the cell death-inducing domains of the protein reside mainly within its amino-terminal two-thirds. The region of BMRP required for the interaction with Bcl-2 is very relevant for the cell death-inducing activity of the protein, suggesting that one possible mechanism by which BMRP induces cell death is by binding to and blocking the anti-apoptotic activity of Bcl-2.